The Role Of Pyruvate In Regulation Of Aging Of Postovulatory Mouse Oocytes

Postovulatory aging of oocytes significantly affects embryonic development. Fertilization of postovulatory aged oocytes gives rise to mice suffering from nervous and emotional abnormalities and decreased reproductive fitness and longevity Like detention in the oviduct, in vitro culture of matured oocytes also led to oocyte aging. However, human beings and some animals potentially undertake sexual activity on any day of the estrous cycle, which may cause fertilization of aged ovulated oocytes. In addition, many experimental designs in both research and clinical applications involve culture of matured oocytes prior to micromanipulation or insemination. Therefore, studies on the mechanisms and control of oocyte aging are important for the healthy reproduction of both human beings and nonhuman mammals.The most prominent manifestations of aged oocytes include an increased susceptibility to activating stimuli, a decrease in MPF activity, the onset of anaphase II, a partial exocytosis of cortical granules, mitochondria aggregation, changes in microtubule assembly and signs of apoptotic cell death such as increased cytoplasmic and DNA fragmentation and decreased levels of Bcl-2 proteins. In addition, an increased acetylation on some lysines of histones and intracellular levels of ROS was increased for the reason of oxidative stress during aging process, subsequently decreased the level of total GSH and the ratio of GSH/GSSG. Our previous studies showed that cumulus cells accelerated aging of mouse oocytes.However, the exact mechanism by which cumulus cells promote oocyte aging is unknown. Since it was reported that matured oocytes were capable of metabolizing pyruvate and that cumulus-oocyte complexes (COCs) consumed much more pyruvate than cumulus-denuded oocytes (DOs) in the absence of glucose, we hypothesized that oocytes would age quickly in the absence of pyruvate, and cumulus cells would accelerate oocyte aging by depleting pyruvate in the CZB medium that contains no glucose. The objective of this study was to test this hypothesis. Newly ovulated mouse oocytes were cultured in the CZB medium supplemented with different concentrations of pyruvate before treated for activation or assayed for MPF activity. While pyruvate decreased the susceptibility to activation stimulus while increased the MPF activity of both COCs and DOs in a dose-dependent manner, more pyruvate was needed in the presence than in absence of cumulus cells. When newly ovulated DOs were cultured in COCs-conditioned CZB without pyruvate supplementation, activation rates increased while MPF activity decreased significantly; however, when cultured in conditioned CZB supplemented with 10 mM pyruvate, both activation rates and MPF activity remained close to those of newly ovulated control oocytes. Capillary electrophoresis showed that CZB conditioned with COCs or cumulus cells contained significantly less pyruvate than freshly prepared CZB and CZB conditioned with DOs or without cells. These results have fully born out our hypothesis that cumulus cells accelerate oocyte aging by depleting pyruvate in the culture medium that contains no glucose.In order to detect the effects of pyruvate on the other indicators of oocytes aging, we observed the premature exocytosis of cortical granules, mitochondria distribution, microtuble assembly, levels of Bcl-2 and histone acetylation through immunofluorescence microscopy; Observed the onset of anaphase II by Hoechst 33342 staining. We also detected the effects of pyruvate on the development potential of aging oocytes through in vitro fertilizaion and subsequent embryonic development. This results suggested that pyrvate can significantly inhibit premature exocytosis of cortical granules, mitochondria aggregation and changes in microtuble assembly; and significantly prevented the decreases of levels of Bcl-2, histone acetylation and the rates of cytoplasmic fragmentation, indicated that pyruvate prevented apoptosis of aging mouse oocytes. In addation, the present results also suggested that pyruvate can significantly weaken the detrimental effects of postovulatory aging on the developmental potential of oocytes. This data confirmed that pyruvate inhibited aging of both COCs and DOs.In order to verify whether the pyruvate inhibited the aging of oocytes through its antioxidative ability we evaluated the intracellular level of ROS through 2 ', 7'-dichlorodihydrofluorescein diacetate (DCHFDA) staining method; and assaied the levels of total GSH, reduced GSH and the ratio of GSH/GSSG through DTNB-GSSG Reductase experiment. The results showed that pyruvate could significantly prevent the increase in the level of ROS, and the decrease of levels of total GSH and reduced GSH and the ratio of GSH/GSSG. It is concluded that pyruvate could maintain the redox state of the aging oocytes and significantly weaken the injury of oocytes resulted from oxidative stress during postovulatory aging.Therefore, we conclude that pyruvate could inhibit the aging of both COCs and DOs, cumulus cells accelerate oocyte aging by depleting pyruvate in the culture medium that contains no glucose. And from the data presented, it is apparent that the supplementation of pyruvate during oocytes aging had several beneficial effects on the qualities of postovulated oocytes and reduced multiple measures of apoptosis. One beneficial effect of pyruvate in preventing the apoptosis is the favorable maintenance of the oocytes GSH-to GSSG ratio and prevention of a decrease in total glutathione content and the increase in ROS maybe through its antioxidative activites.